RESUMO
The dissociative anesthetic ketamine regulates cortical activity in a dose-dependent manner. Subanesthetic-dose ketamine has paradoxical excitatory effects which is proposed to facilitate brain-derived neurotrophic factor (BDNF) (a ligand of tropomyosin receptor kinase B, TrkB) signaling, and activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Previous data suggests that ketamine, at sub-micromolar concentrations, induces glutamatergic activity, BDNF release, and activation of ERK1/2 also on primary cortical neurons. We combined western blot analysis with multiwell-microelectrode array (mw-MEA) measurements to examine ketamine's concentration-dependent effects on network-level electrophysiological responses and TrkB-ERK1/2 phosphorylation in rat cortical cultures at 14 days in vitro. Ketamine did not cause an increase in neuronal network activity at sub-micromolar concentrations, but instead a decrease in spiking that was evident already at 500 nM concentration. TrkB phosphorylation was unaffected by the low concentrations, although BDNF elicited prominent phosphorylation response. High concentration of ketamine (10 µM) strongly reduced spiking, bursting and burst duration, which was accompanied with decreased phosphorylation of ERK1/2 but not TrkB. Notably, robust increases in spiking and bursting activity could be produced with carbachol, while it did not affect phosphorylation of TrkB or ERK1/2. Diazepam abolished neuronal activity, which was accompanied by reduced ERK1/2 phosphorylation without change on TrkB. In conclusion, sub-micromolar ketamine concentrations did not cause an increase in neuronal network activity or TrkB-ERK1/2 phosphorylation in cortical neuron cultures that readily respond to exogenously applied BDNF. Instead, pharmacological inhibition of network activity can be readily observed with high concentration of ketamine and it is associated with reduced ERK1/2 phosphorylation.
Assuntos
Ketamina , Ratos , Animais , Ketamina/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Neurônios , Fosforilação , Receptor trkB/metabolismoRESUMO
Genetic diversity was studied in a collection of 61 accessions of Brassica rapa, which were mostly oil-type turnip rapes but also included two oil-type subsp. dichotoma and five subsp. trilocularis accessions, as well as three leaf-type subspecies (subsp. japonica, pekinensis, and chinensis) and five turnip cultivars (subsp. rapa). Two-hundred and nine SNP markers, which had been discovered by amplicon resequencing, were used to genotype 893 plants from the B. rapa collection using Illumina BeadXpress. There was great variation in the diversity indices between accessions. With STRUCTURE analysis, the plant collection could be divided into three groups that seemed to correspond to morphotype and flowering habit but not to geography. According to AMOVA analysis, 65% of the variation was due to variation within accessions, 25% among accessions, and 10% among groups. A smaller subset of the plant collection, 12 accessions, was also studied with 5727 GBS-SNPs. Diversity indices obtained with GBS-SNPs correlated well with those obtained with Illumina BeadXpress SNPs. The developed SNP markers have already been used and will be used in future plant breeding programs as well as in mapping and diversity studies.
Assuntos
Brassica rapa/genética , Polimorfismo de Nucleotídeo Único , Brassica napus/genética , Brassica rapa/metabolismo , Ácidos Graxos/metabolismo , Variação Genética , Glucosinolatos/metabolismo , Filogeografia , Melhoramento Vegetal , Doenças das Plantas/genéticaRESUMO
Cross-linked and quaternized pine sawdust was tested for vanadium removal from a synthetic aqueous solution as well as from real industrial wastewater which had a considerable amount of vanadium and other ions such as sulphate, ammonium and nickel. The maximum vanadium sorption capacity of the modified pine sawdust was found to be 130 mg/g in synthetic solution and 103 mg/g in real wastewater. Modified pine sawdust worked well over a wide range of pH. Column studies with real wastewater proved that vanadium was efficiently desorbed from the material with 2 M NaOH and that the material could be reused.
Assuntos
Pinus , Vanádio/química , Eliminação de Resíduos Líquidos/métodos , Águas Residuárias/química , Poluentes Químicos da Água/química , Madeira/química , Adsorção , Purificação da Água/métodosRESUMO
An evaluation was made of the performance of a commercial industrial-scale ultrafiltration (UF)-based process for treatment of highly concentrated oily wastewaters. Wastewater samples were gathered from two plants treating industrial wastewaters in 2008, and in 2011 (only from one of the plants), from three points of a UF-based treatment train. The wastewater samples were analyzed by measuring the BOD7, COD, TOC and total surface charge (TSC). The inorganic content and zeta potentials of the samples were analyzed and GC-FID/MS analyses were performed. The removal performances of BOD7, COD, TOC and TSC in 2008 and 2011 for both plants were very high. Initial concentrations of contaminants in 2011 were lower than in 2008, therefore the COD and TSC reductions were also lower in 2011 than three years before. Regardless of the high performance of UF-based processes in both plants, at times the residual concentrations were considerable. This could be explained by the high initial concentrations and also by the presence of the dissolved compounds that were characterized. Linear correlation was observed between COD and TOC, and between COD and TSC. The correlation between COD and TSC could be utilized for process control purposes.
Assuntos
Ultrafiltração , Eliminação de Resíduos Líquidos/métodos , Biodegradação Ambiental , Análise da Demanda Biológica de Oxigênio , Carbono/análise , Finlândia , Concentração de Íons de Hidrogênio , Resíduos Industriais , Óleos , Águas Residuárias/análiseRESUMO
The GafD lectin of the G (F17) fimbriae of diarrhea-associated Escherichia coli was overexpressed and purified from the periplasm of E. coli by affinity chromatography on GlcNAc-agarose. The predicted mature GafD peptide comprises 321 amino acids, but the predominant form of GafD recovered from the periplasm was 19,092 Da in size and corresponded to the 178 N-terminal amino acid residues, as judged by mass spectrometry and amino acid sequencing, and was named DeltaGafD. Expression of gafD from the cloned gaf gene cluster in DegP-, Lon-, and OmpT-deficient recombinant strains did not significantly decrease the formation of DeltaGafD. The peptide was also detected in the periplasm of the wild-type E. coli strain from which the gaf gene cluster originally was cloned. We expressed gafD fragments encoding C-terminally truncated peptides. Peptides GafD1-252, GafD1-224, GafD1-189, and the GafD1-178, isolated from the periplasm by affinity chromatography, had apparent sizes closely similar to that of DeltaGafD. Only trace amounts of truncated forms with expected molecular sizes were detected in spheroplasts. In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but not in the periplasm, indicating that it was poorly translocated or was degraded by periplasmic proteases. Pulse-chase assays using gafD indicated that DeltaGafD was processed from GafD and is not a primary translation product. The DeltaGafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose. Inhibition assays with mono- and oligosaccharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant E. coli strain and in the binding of [(14)C]DeltaGafD to GlcNAc-agarose. DeltaGafD bound specifically to laminin, a previously described tissue target for the G fimbria. Our results show that a soluble, protease-resistant subdomain of GafD exhibits receptor-binding specificity similar to that for intact G fimbriae and that it is formed when gafD is expressed alone or from the gaf gene cluster.
Assuntos
Adesinas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Fímbrias , Fímbrias Bacterianas/genética , Genes Bacterianos , Lectinas/genética , Família Multigênica , Acetilglucosamina/metabolismo , Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos , Especificidade de Anticorpos , Compartimento Celular , Escherichia coli/patogenicidade , Lectinas/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos , Periplasma , Proteínas Recombinantes de Fusão , Solubilidade , Esferoplastos , Virulência/genéticaRESUMO
The efficiencies of four different waste collection methods were compared by modelling the collection of source-separated household waste in the Helsinki region, Finland. Efficiency was assessed on the basis of system costs, fuel consumption, working hours and recovery rates. The study included paper, biowaste, energy waste and mixed waste produced in households. Both separation of new types of recyclables and increasing the coverage of separate collection increased the costs of household waste collection independently of the collection method studied. Methods in which several types of waste are collected simultaneously increased the efficiency of waste collection. High participation rates and separation efficiencies reduced the costs of waste collection.
Assuntos
Conservação dos Recursos Naturais/métodos , Produtos Domésticos , Eliminação de Resíduos/métodos , Conservação dos Recursos Naturais/economia , Custos e Análise de Custo , Eliminação de Resíduos/economiaRESUMO
A multivalent, bifunctional flagellum carrying two different adhesive peptides in separate flagellin subunits within a filament was constructed in Escherichia coli. The inserted peptides were the fibronectin-binding 115-mer D repeat region of Staphylococcus aureus and the 302-mer collagen-binding region of YadA of Yersinia enterocolitica. Western blotting, immunoelectron microscopy, and adhesion tests with hybrid flagella from an in trans-complemented DeltafliC E. coli strain showed that individual filaments consisted of both recombinant flagellins.
Assuntos
Adesinas Bacterianas/genética , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Escherichia coli/metabolismo , Flagelos/metabolismo , Flagelina/genética , Yersinia enterocolitica/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Escherichia coli/genética , Flagelina/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Yersinia enterocolitica/química , Yersinia enterocolitica/genéticaRESUMO
The replicative spread of retrotransposons in the genome creates new insertional polymorphisms, increasing retrotransposon numbers and potentially both their share of the genome and genome size. The BARE-1 retrotransposon constitutes a major, dispersed, active component of Hordeum genomes, and BARE-1 number is positively correlated with genome size. We have examined genome size and BARE-1 insertion patterns and number in wild barley, Hordeum spontaneum, in Evolution Canyon, Lower Nahal Oren, Mount Carmel, Israel, along a transect presenting sharply differing microclimates. BARE-1 has been sufficiently active for its insertional pattern to resolve individuals in a way consonant with their ecogeographical distribution in the canyon and to distinguish them from provenances outside the canyon. On both slopes, but especially on the drier south-facing slope, a simultaneous increase in the BARE-1 copy number and a decrease in the relative number lost through recombination, as measured by the abundance of solo long terminal repeats, appear to have driven the BARE-1 share of the genome upward with the height and dryness of the slope. The lower recombinational loss would favor maintenance of more full-length copies, enhancing the ability of the BARE-1 family to contribute to genome size growth. These local data are consistent with regional trends for BARE-1 in H. spontaneum across Israel and therefore may reflect adaptive selection for increasing genome size through retrotransposon activity.
Assuntos
Evolução Biológica , Genes de Plantas , Hordeum/genética , Retroelementos , Citometria de Fluxo , Dosagem de Genes , Sequências Repetidas TerminaisRESUMO
The replicative retrotransposon life cycle offers the potential for explosive increases in copy number and consequent inflation of genome size. The BARE-1 retrotransposon family of barley is conserved, disperse, and transcriptionally active. To assess the role of BARE-1 in genome evolution, we determined the copy number of its integrase, its reverse transcriptase, and its long terminal repeat (LTR) domains throughout the genus Hordeum. On average, BARE-1 contributes 13.7 x 10(3) full-length copies, amounting to 2.9% of the genome. The number increases with genome size. Two LTRs are associated with each internal domain in intact retrotransposons, but surprisingly, BARE-1 LTRs were considerably more prevalent than would be expected from the numbers of intact elements. The excess in LTRs increases as both genome size and BARE-1 genomic fraction decrease. Intrachromosomal homologous recombination between LTRs could explain the excess, removing BARE-1 elements and leaving behind solo LTRs, thereby reducing the complement of functional retrotransposons in the genome and providing at least a partial "return ticket from genomic obesity."
RESUMO
Propagation of long terminal repeat (LTR)-bearing retrotransposons and retroviruses requires integrase (IN, EC 2.7.7.-), encoded by the retroelements themselves, which mediates the insertion of cDNA copies back into the genome. An active retrotransposon family, BARE-1, comprises approximately 7% of the barley (Hordeum vulgare subsp. vulgare) genome. We have generated models for the secondary and tertiary structure of BARE-1 IN and demonstrate their similarity to structures for human immunodeficiency virus 1 and avian sarcoma virus INs. The IN core domains were compared for 80 clones from 28 Hordeum accessions representative of the diversity of the genus. Based on the structural model, variations in the predicted, aligned translations from these clones would have minimal structural and functional effects on the encoded enzymes. This indicates that Hordeum retrotransposon IN has been under purifying selection to maintain a structure typical of retroviral INs. These represent the first such analyses for plant INs.
Assuntos
Evolução Molecular , Hordeum/enzimologia , Integrases/genética , Retroelementos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Hordeum/genética , Integrases/química , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Retrotransposons propagate via an RNA intermediate which is then reverse-transcribed and packaged into virus-like particles. They are either copia- or gypsy-like in coding domain order and sequence similarity, the gypsy-like elements sharing their organization with the retroviruses but lacking retroviral envelope domains. Copia-like retrotransposons, or at least their reverse transcriptase domains, appear broadly distributed in higher plants, but gypsy-like elements have been reported only for scattered species. The authors have exploited the difference in domain order between these groups to amplify and clone segments bridging the reverse transcriptase-integrase region of specifically gypsy-like retrotransposons. Species representative of the diversity of higher plants yielded products whose sequences establish that gypsy-like transposons are dispersed throughout the plant genomes. This class of plant elements has been named romani retrotransposons. The presence of both types ubiquitously in the fungi, plants and animals support their existence as ancient distinct lineages and subsequent, vertical radiation.
Assuntos
Plantas/genética , Retroelementos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Plantas/classificação , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
Presents a novel neural-network-based predictor for received power level prediction in direct sequence code division multiple access (DS/CDMA) systems. The predictor consists of an adaptive linear element (Adaline) followed by a multilayer perceptron (MLP). An important but difficult problem in designing such a cascade predictor is to determine the complexity of the networks. We solve this problem by using the predictive minimum description length (PMDL) principle to select the optimal numbers of input and hidden nodes. This approach results in a predictor with both good noise attenuation and excellent generalization capability. The optimized neural networks are used for predictive filtering of very noisy Rayleigh fading signals with 1.8 GHz carrier frequency. Our results show that the optimal neural predictor can provide smoothed in-phase and quadrature signals with signal-to-noise ratio (SNR) gains of about 12 and 7 dB at the urban mobile speeds of 5 and 50 km/h, respectively. The corresponding power signal SNR gains are about 11 and 5 dB. Therefore, the neural predictor is well suitable for power control applications where ldquodelaylessrdquo noise attenuation and efficient reduction of fast fading are required.
RESUMO
An expression system for studying epitopes of adhesion proteins based on fusion of gene fragments into fliC(H7) of Escherichia coli is described. We constructed the system by an in-frame insertion of DNA fragments encoding one, two or three of the fibronectin-binding D repeats present in the fibronectin-binding protein A (FnBPA) of Staphylococcus aureus, into the fliC(H7) gene region encoding the variable domain of the H7 flagellin. The constructs were expressed by in trans complementation in the E. coli strain JT1 which harbours knock-out mutations for the expression of FliC as well as of the mannoside-binding fimbrial adhesin. The resulting chimeric flagella, which contained 39, 77 or 115 heterologous amino acid residues, efficiently bound soluble and immobilized human plasma and cellular fibronectin, and the binding was most efficient with the flagella containing the three D repeats of FnBPA. The chimeric flagella bound to frozen sections of human kidney and to cultured human cells. Antibodies raised against the chimeric flagella bound to Protein A-deficient S. aureus cells and inhibited the binding of staphylococci to immobilized fibronectin. We also expressed peptides, ranging in size between 48 and 302 amino acids, of the collagen-binding YadA adhesin of Yersinia enterocolitica. A fragment of 302 amino acids representing the middle region of YadA was needed for collagen binding. Chimeric flagellar filaments expressing hundreds of intimately associated adhesive epitopes offer versatile tools to analyze adhesin-receptor interactions and functional epitopes of adhesion proteins.